Strawberry Culture In Vitro: Applications in Genetic Transformation and Biotechnology

Transcription

1 Fruit, Vegetable and Cereal Science and Biotechnology 2007 Global Science Books Strawberry Culture In Vitro: Applications in Genetic Transformation and Biotechnology Samir C. Debnath 1*,** Jaime A. Teixeira da Silva 2 1 Atlantic Cool Climate Crop Research Centre, Agriculture and Agri-Food Canada, P.O. Box 39088, 308 Brookfield Road, St. John's, Newfoundland and Labrador A1E 5Y7, Canada 2 Kagawa University, Faculty of Agriculture, Department of Horticulture, Miki cho, Ikenobe, , Kagawa ken, Japan Corresponding author: * debnaths@agr.gc.ca ABSTRACT The cultivated strawberry (Fragaria ananassa Duch.), a member of the Rosaceae, is the most important soft fruit worldwide. In vitro techniques are important for clonal multiplication, germplasm improvement and for gene conservation of this flavourful and nutritious berry crop. The in vitro propagation of Fragaria species using axillary bud proliferation, adventitious shoot regeneration and somatic embryogenesis has been investigated in a number of previous studies. The morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. In strawberry, genetic transformation has been developed using tissue culture systems with varying rates of success. This review presents the progress in-depth of various aspects of strawberry culture in vitro, on gelled and in liquid media using bioreactors, for its improvement and for commercial production. It also discusses the issues that still need to be addressed to utilize the full potential of plant tissue culture techniques in mass propagation, in vitro selection, somaclonal variation, haploid recovery, somatic hybridization, genetic transformation and in cryopreservation of strawberries. Application of molecular marker techniques should be useful to verify the clonal fidelity of micropropagated strawberries. Strawberry improvement using in vitro and molecular techniques will develop improved cultivars suited to the changing needs of growers and consumers. Keywords: micropropagation, regeneration, somaclonal variation CONTENTS INTRODUCTION... 1 MICROPROPAGATION... 2 Shoot proliferation and virus elimination... 2 Adventitious shoot regeneration... 3 Protoplast culture and somatic hybridization... 3 Anther culture and haploid recovery... 4 Rooting and acclimatization... 4 Somatic embryogenesis... 5 Bioreactor micropropagation... 5 Field evaluation of micropropagated plants... 5 SOMACLONAL VARIATION... 6 IN VITRO SELECTION... 6 GENETIC TRANSFORMATION... 6 LOW-TEMPERATURE STORAGE AND CRYOPRESERVATION... 8 CONCLUDING REMARKS... 8 REFERENCES... 9 ** This work is the Atlantic Cool Climate Crop Research Centre contribution no INTRODUCTION The cultivated strawberry (Fragaria ananassa Duch.), a hybrid between the Scarlet or Virginia strawberry (F. virginiana Duch.) and the pistillate South American F. chiloensis (L.) Duch., is a dicotyledonous, perennial low-growing herb grown in most arable regions of the World. There are about 20 recognized species of strawberries in five chromosome groups (x = 7): ten diploids, four tetraploids, one pentaploid, one hexaploid and four octoploids (Staudt 1999; Jiajun et al. 2005). The cultivated strawberry is an octoploid (2n = 8x = 56). Flavourful and nutritious, strawberries are enjoyed by millions of people in all climates, including temperate, Mediterranean, sub-tropical and taiga zones (Hancock et al. 1991) and are predominantly used as fresh fruit. Their use in processed forms such as cooked and sweetened preserves, jams or jellies and frozen whole berries or sweetened juice extracts or flavorings, and their use in making a variety of other processed products made them one of the most popular berry crops, more widely distributed than any other fruit (Childers 1980). The berry is valued for its low-calorie carbohydrate and high fiber contents. Strawberries are good sources of natural antioxidants (Wang et al. 1996; Heinonen et al. 1998) including carotenoids, vitamins, phenols, flavonoids, dietary glutathionine, and endogenous metabolites (Larson 1988) and exhibit a high level of antioxidant capacity against free radical species: superoxide radicals, hydrogen peroxide, hydroxyl radicals and singlet oxygen (Wang and Jiao 2000). Meyers et al. (2003) showed that phenolics Received: 5 February, Accepted: 2 April, Special Feature

2 Fruit, Vegetable and Cereal Science and Biotechnology 1(1), Global Science Books in strawberries account for a major portion of the total antioxidant activity of strawberries. Strawberry extracts were found to have higher antioxidant activity, as indicated by the oxygen radical absorbance capacity assay, than extracts from plum, orange, red grape, kiwifruit, pink grapefruit, white grape, banana, apple, tomato, pear and honeydew melon (Wang et al. 1996). Similarly, Sun et al. (2002), using a total antioxidant oxyradical scavenging assay (TOSC), found that strawberry extracts had higher antioxidant activity than extracts from peach, lemon, banana, pear, orange, grapefruit and pineapple. The benefits of these high antioxidant activity fruit include reduction of carcinogens in humans (Chung et al. 2002), protection against tumor development (Kresty et al. 2001) and reversal of age-related effects on memory (Bickford et al. 2000). Strawberry antioxidant activity levels are affected not only by the genotype but also by both growing temperatures (Wang and Zheng 2001) and cultural practices (Wang et al. 2002). In vitro techniques are important tools for modern plant improvement programs to introduce new traits into selected plants, to multiply elite selections and to develop suitable cultivars in the minimum time (Taji et al. 2002). Used in conjunction with classical breeding methods, an efficient in vitro shoot proliferation and regeneration system could accelerate cultivar development programs. The ability to regenerate plants is crucial to the successful application of in vitro methods (Cao and Hammerschlag 2000). A shoot regeneration system can be used to develop transgenic plants following genetic transformation of plant cells and to identify and/or induce somaclonal variants. MICROPROPAGATION The totipotency exhibited by the apical meristem and the adjacent shoot tip region is the cornerstone for commercial micropropagation. Micropropagation of strawberry plants was introduced about thirty years ago (Boxus 1974). Immediately, the most important European nurseries producing several millions plants per year, were interested in this technique as it gave a definitive answer to the problems of soil fungi, causing a lot of damage to the strawberry fields and by another way, tissue culture plants seemed to produce more runners per mother plant in a short time (Mohan et al. 2005). Micropropagation has also been widely used in the USA (Zimmerman 1981) in commercial propagation of strawberries and in breeding programs to produce many plants rapidly. Conventionally, strawberries are vegetatively propagated by runners arising from axillary leaf buds on the plant crown. Plant propagation through runner produces a limited number of propagules. Although production of propagules through runner has been reported to contribute 90% of total Dutch strawberry production, the product in Elsanta cultivar was found to be susceptible to several fungal diseases (Dijkstra 1993). Micropropagated strawberry plants can be stored under refrigeration (Mullin and Schlegel 1976), making it a valuable technique for storage of germplasm. Complete new plants can be derived from tissue either from pre-existing buds through shoot proliferation, following shoot morphogenesis through adventitious shoot regeneration or through the formation of somatic embryos. Micropropagation differs from all other conventional propagation methods in that aseptic conditions are essential to achieve success. Shoot proliferation and virus elimination Plants produced by axillary branching normally retain the genetic composition of the mother plant and this method has proven to be the most applied and reliable method for true-to-type in vitro propagation in general. Successful shoot proliferation has been obtained in strawberry from single meristems (Boxus 1974), meristem callus (Nishi and Oosawa 1973) and from node culture (Bhatt and Dhar 2000). Morel (1960) was the pioneer for meristem culture. Meristem-tip culture alone or in combination with heat treatment (Yoshino and Hashimoto 1975) is widely used to obtain virus and fungus-free strawberry plants (Molot et al. 1972). Posnette (1953) originally developed the technique of hot air therapy to eliminate viruses, and Belkengren and Miller (1962) began the practice of excising heat-treated meristems for placement on tissue culture media. Potted plants are placed in a growth chamber at ambient temperatures and then this temperature is raised a few degrees a day up to 38 C and grown for 6 weeks (Lines et al. 2006). Shoot tips are removed after treatment and the meristem, a dome of actively dividing cells, about 0.1 mm in diameter and 0.25 mm long, with two or three leaf primordia are removed and cultured on a nutrient medium. Most commonly used explant for strawberry micropropagation is the meristem from the tip of runners (Sowik et al. 2001). The explant is placed on a medium containing no or low levels of auxins and higher levels of cytokinins to promote axillary budding while preventing callus formation. The cytokinins are used to overcome apical dominance and enhance the branching of lateral buds from the leaf axis. Additional shoots are produced through further axillary bud growth (Debnath 2003). Mullin et al. (1974) grew strawberries with strawberry mild yellow edge (SMYE) viruses for 6 weeks in a 36 C growth chamber before excising 0.3 to 0.8 mm meristematic tips with leaf primordia. The result was that 33% to 75% of the resulting plants were SMYE-free according to leaf insert graft indexing to indicator strawberry plants. In fact, Mullin et al. (1974) maintained strawberries that had received heat therapy and were propagated using apical meristematic regions, free from detectable graft transmissible diseases for seven years in a greenhouse and a screenhouse. Cultures can be initiated and maintained on Boxus (Boxus 1974) medium containing Knop s (Knop 1965) macronutrients and Murashige and Skoog (1962, MS) micronutrients and organic components, or MS medium supplemented with µm 6-benzyladenine (BA), µm indole- 3-butyric acid (IBA) and 0.3 µm gibberellic acid (GA 3 ) (Cerović and Ružić 1989) at C during the light period, and 17 C in the dark; the quantum irradiance is 46 µmol m -2 s -1 for a 16 h photoperiod (Sowik et al. 2001). Runners can be initiated from in vitro culture on media containing AgNO 3 (10-20 mg/l). GA 3 ( µm) increased the efficiency of AgNO 3 significantly (Zatykó et al. 1989). Borkowska (2001) initiated strawberry cultures according to Boxus (1974, 1992), but modified medium by lowering the concentration of cytokinin (BA, 2.2 µm) and auxin (IBA, 0.5 µm). Although agar ( %, w/v) is the most commonly used gelling agent for in vitro strawberry culture on semi-solid medium, Lucyszyn et al. (2006) reported that the agar/galactomannan (gour, Indian Gum Industries, Jodhpur, India) mixture in the proportion of 0.3/0.3 (w/v) in MS medium showed better performance and enhanced shoot proliferation compared to medium containing agar (0.6%, w/v) only. Cultures were maintained at 23 C under a photosynthetic photon flux density (PPFD) of 30 µmol m -2 s -1 from warm-white fluorescent lamps and 16 h photoperiod. The use of light-emitting diodes, or LEDs, in particular 70% red and 30% blue at 60 µmol m -2 s -1 resulted in greatest shoot proliferation, plantlet and total fresh weight when three-leaved explants of cv. Akihime derived from conventional mixotrophic cultures were placed in a photoautotrophic culture vessel, the Culture Pack, using rockwool as a substrate, 0.2. mg/l BA in MS basal medium and with CO 2 -enrichment (3000 ppm) (Nhut et al. 2000, 2003). Plantlets from the same treatment also resulted in 100% acclimatization. Similar results could be obtained in a much more simplified system using conventional 3.6 l polyethylene vessels, in which one culture vessel was estimated to cost less than 1 $US, and in which up to strawberry plantlets could be developed and rooted from three-leaved (rootless) explants (Nhut et al. 2006). In this system the authors found that the use of Micropore TM 3M, added at 4 sheets (approx 2 cm 2 ) per box, 2

3 Strawberry culture in vitro. Debnath and Teixeira da Silva would increase the ventilation to the vessels and enhance the development of shoots and their subsequent acclimatization. Bhatt and Dhar (2000) micropropagated wild strawberry (F. indica Andr.) using node culture. Nodal segments (2.5-3 cm), treated with Savlon (an antiseptic containing 3% antimicrobial agent centrimide + detergent) for 15 min, were immersed in 80% ethanol for 30 sec prior to surface sterilization in HgCl 2 (0.05%) containing few drops of Tween 20 for 5 min and then cultured on MS medium supplemented with 4 µm BA and 0.1 µm α-naphthalene acetic acid (NAA). The same medium was used for shoot multiplication. Adventitious shoot regeneration There have been a number of reports for adventitious bud and shoot regeneration from leaves (Nehra et al. 1988; Jones et al. 1988; Lui and Sandford 1988; Nehra et al. 1989, Sorvari et al. 1993; Passey et al 2003; Debnath 2005; Qin et al. 2005a, 2005b; Yonghua et al. 2005; Debnath 2006), petioles (Foucault and Letouze 1987; Rugini and Orlando 1992; Passey et al. 2003; Debnath 2005, 2006), peduncles/peduncular base of the flower bud (Foucault and Letouze 1987; Lis 1993), stems (Graham et al. 1995), stipules (Rugini and Orlando 1992; Passey et al. 2003), stolons (Lis 1993), roots (Rugini and Orlando 1992; Passey et al. 2003), runners (Liu and Sanford 1988), mesophyll protoplasts (Nyman and Wallin 1988), anther cultures (Owen and Miller 1996) and from immature embryos (Wang et al. 1984) of strawberries. Shoot regeneration directly from field- (Nehra et al. 1989) or greenhouse-grown strawberry plants (Barceló et al. 1998; Debnath 2005, 2006) or from in vitro-grown shoots (Sovari et al. 1993; Passey et al. 2003; Yonghua et al. 2005) have been reported. Explants taken from field-grown plants are difficult to sterilize to establish in vitro cultures due to high degree of contamination. It is usually recommended to take explants from plants grown under controlled conditions such as growth room or greenhouse, or from buds which flush from dormant shoots stored indoors. Plant regeneration is a crucial aspect of plant biotechnology methodology and tissue culture that facilitates the production of genetically engineered plants and somaclonal variants, and the rapid multiplication of difficult-to-propagate species. Adventitious plant regeneration from strawberry explants can be divided into the following steps: (i) formation of viable adventitious buds on the explant; (ii) elongation of the buds into shoots; (iii) rooting of the shoots to form whole plants. A number of factors such as genotype, culture medium (including growth regulators and their combinations) (Table 1), physical environment, explant development stage, etc. affect adventitious shoot regeneration. Thidiazuron (TDZ), a the substituted phenylurea (Nphenyl-N'-1,2,3-thidiazol-5-ylurea) with its cytokinin- and auxin-like effects, is now among the most active cytokininlike substances for plant tissue culture and has been used to induce shoot organogenesis of strawberries (Table 1). TDZ alone (Debnath 2005) or in combination with 2,4-dichlorophenoxy-acetic acid (2,4-D) (Passey et al. 2003) or IBA (Yonghua et al. 2005) was found to be effective for shoot regeneration from strawberry leaves. While leaf explants were used in most of the early studies for bud and shoot regeneration, sepals (Table 1), a floral leaf or individual segment of the calyx of a flower that forms the outer protective layer in a bud, have been tested for shoot regeneration of in vitro strawberry cultures (Debnath 2005). Shoot regeneration was obtained from sepal, leaf and petiole explants by incorporating TDZ (2-4 μm) in the culture medium and a dark treatment for 14 d before incubating the explants under a 16-h photoperiod. A dark treatment similar to those used for strawberry (Barceló et al. 1998) leaf generation was used to achieve the highest response. Such TDZinduced shoots were transferred to 2-4 μm zeatin-containing medium for elongation (Debnath 2005). Callus regeneration and shoot formation depended not only on the explant orientation and polarity, but also on genotype (Passey et al. 2003). Young expanding sepals with the adaxial side touching the culture medium produced the best results. Qin et al. (2005b) reported that Toyonoka strawberry leaf explants cultured for 10 days in shoot regeneration medium in the presence of AgNO 3 not only enhanced shoot regeneration efficiency but also expedited the inhibition of adventitious buds. Being an ethylene inhibitor, AgNO 3 can markedly promote organogenesis in strawberries. Protoplast culture and somatic hybridization The plant protoplast, a naked cell, consisting of the cytoplasm and nucleus with the cell wall removed, provides a unique single cell system to underpin several aspects of Table 1 Examples of basal media 1 and plant growth regulators (PGR, mg/l) 2 used for adventitious shoot regeneration of strawberries in vitro. Cultivar Explant source Basal medium (mg/l) Shoot regeneration Reference Redcoat, Veestar, Bounty, Leaf disk MS salts + B-5 vitamins 2.3 BA IAA Nehra et al Kent, Micmac, Glooscap, Honeoye, Hecker, Fern, Selva Hiku, Jonsok Leaf disk MS salts + 39 Fe(III)Na-EDTA 3 BA IBA Sorvari et al KNO CH Chandler Leaf disk N 30K macrosalts + MS 2 BA IBA Barceló et al microsalts and vitamins Calypso, Pegasus, Bolero, Leaf disk, petiole, MS 1 TDZ D, 2 BA Passey et al Tango, Elsanta root, stipule TDZ ,4-D, 1 TDZ NAA or 2 BA ,4-D Sweet Charlie, Pajaro Leaf disk ½MS 1 BA + 1 IAA Singh and Pandey 2004 Senga Sengana Leaf petiole Boxus (1974) 0.5 BA GA IBA Litwińczuk 2004 Hecker, La Sans Rivale, diploid Leaf, petiole MS salts + B5 vitamins 2.2 TDZ IBA Zhao et al accessions (FRA197, FRA198) Bounty Sepal, leaf disk, BM-D TDZ Debnath 2005 petiole half Toyonoka Leaf disk MS 1.5 TDZ IBA Qin et al. 2005a Toyonoka Leaf disk MS + 1 AgNO 3 1.5TDZ IBA Qin et al. 2005b Bounty, Jonsok, Korona, Polka, Zephyr Leaf MS 2 TDZ IBA Hanhineva et al LF9 Leaf, petiole, stolon MS 0.11 BA ,4-D + 1 TDZ Folta et al Media: BM-D = Debnath and McRae (2001); MS = Murashige and Skoog (1962); N 30K = Margara (1984). 2 PGR: 2,4-D = 2,4-dichlorophenoxy-acetic acid, BA = 6-benzyladenine, GA 3 = gibberellic acid, IBA = indole-3-butyric acid, IAA = 3-indolyl-acetic acid, NAA = α- naphthalene acetic acid, TDZ = thidiazuron. 3

4 Fruit, Vegetable and Cereal Science and Biotechnology 1(1), Global Science Books modern biotechnology (Davey et al. 2005). Although reliable procedures are available to isolate and culture protoplasts from a range of plants (Davey et al. 2005), there have been few reports in strawberry. Fong et al. (1889) had limited success for protoplast isolation but failed to culture them. The need for protoplast technology for gene transfer, somatic hybridization and for somaclonal variation (Larkin and Scowcroft 1981) in strawberry breeding has been indicated by several authors (Wallin et al. 1993). Leaf- and petiole-derived protoplasts (Nyman and Wallin 1988, 1992) and protoplasts from callus (Wallin 1997) have been isolated and cultured with a protoplasts/ g fresh weight of viable protoplasts yield (Nyman and Wallin 1992). Several parameters, particularly the source tissue, culture medium and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Protoplasts can be isolated mechanically by cutting or breaking the cell wall, and by digesting it away with enzymes or by a combination of mechanical and enzymatic separation (George 1993). Isolated strawberry protoplasts can be cultured on 8P medium (Glimelius et al. 1986) with either 1 mg/l 2,4-D and 0.5 mg/l BA (Nyman and Wallin 1992) or 2 mg/l NAA and 0.5 mg/l TDZ (Wallin et al. 1993). Shoot organogenesis is induced on MS medium with 2% sucrose and 2 mg/l BA and 0.2 mg/l NAA. Measurement of the DNA content of these plants has revealed a range of ploidy levels. Infante and Rosati (1993) isolated protoplasts and regenerated plants in wood strawberry (F. vesca). Plants grew normally in the greenhouse and did not show any visible abnormality compared to the original clone multiplied through micropropagation. Protoplasts may be fused together to create plant cell hybrids. Isolated protoplasts do not normally fuse together because they carry a superficial negative charge causing them to repeal one another. The two most successful techniques of protoplast fusion are (i) the addition of polyethylene glycol (PEG) in the presence of a high concentration of calcium ions and a ph between 8-10 and (ii) the application of short pulses of direct electrical current (electro-fusion). Somatic hybridization offers the possibility of genetic exchange between the diploid F. vesca with cultivated octoploid strawberry. Wallin (1997) regenerated plants from calli originating from protoplast fusion between F. ananassa and F. vesca. Protoplasts of F. ananassa resistant to hygromycin were also fused to protoplasts of F. ananassa resistant to kanamycin. Protoplast fusion-derived plants that deviated morphologically from the parents had >56 chromosomes which might be due to somaclonal variation (Wallin 1997). Anther culture and haploid recovery Anther culture involves the aseptic culture of immature anthers to generate fertile haploid plants from microspores. The production of haploid plants through anther culture is widely used for breeding purposes, as an alternative to the numerous cycles of inbreeding or backcrossing usually needed to obtain pure lines in conventional breeding. Chromosome doubling of haploids could result in immediate establishment of homozygosity for all loci. The success achieved with anther culture has led to the development of microspore culture to regenerate homozygous plants. Furthermore, isolated microspores are very attractive for protoplast isolation and applications aiming at transformation as they are unicellular and transgenic homozygous plants could be provided in a comparatively short time (Dhlamini et al. 2005; Germanà 2006). Haploid recovery in strawberry through aseptic anther culture was unsuccessful in some early reports (Rosati et al. 1975), Niemirowicz-Szczytt et al. 1983). Owen and Miller (1996) obtained haploid plants from cultured anthers of Chandler, Honeoye and Redchief strawberries. The highest shoot regeneration across cultivars (8%) was obtained when a semi-solid MS medium contained 2 mg/l IAA, 1 mg/l BA and 0.2 M glucose. Chromosome counts of root tip cells from ex vitro-grown plants confirmed that haploid plants were obtained from all three cultivars. Rooting and acclimatization Both in vitro and ex vitro methods have successfully been used to root and acclimatize micropropagated strawberry shoots. Proliferated shoots can be rooted in vitro on Boxus (Borkowska 2001; Sowik et al. 2001), half-strength MS (Yue et al. 1993) or modified cranberry (Debnath 2005) medium without growth regulators, or on half-strength MS with activated charcoal (0.6 g/l) and IAA (5.7 µm) (Moore et al. 1991). The in vitro-formed roots are thick, posses no hairy roots, grow horizontally, and are fragile and easily damaged. In vitro-grown plantlets have low photosynthetic activity, poor water balance and their anatomy and morphology are far from being optimal (Grout and Millam 1995; Borkowska 2001). Kozai (1991) and Hayashi et al. (1997) developed a photoautotrophic micropropagation system for plant multiplication by simultaneously increasing the CO 2 concentration and light quality. Plantlets that are rooted ex vitro have a larger root system and more runners than those formed by in vitro-rooted strawberry plants (Borkowska 2001). Similar results were observed by Nhut et al. (2000, 2003, 2006) when using either CO 2 -enrichment with or without light-emitting diodes, or even when a simple improved aeration system was implemented in mixotrophic culture. When moved from in vitro culture conditions, microcuttings must be acclimatized gradually to ambient conditions to avoid mortality that might otherwise occur under an abrupt change in relative humidity, temperature or irradiance. For in vitro-rooted plantlets, standard procedure is to wash the plantlets and transfer to pots containing ProMix BX (Premier Horticulture Limited, Riviére-du-Loup, Québec, Canada) (Debnath 2005, 2006) or 1 peat : 1 vermiculite (Zhou et al. 2005), and maintained in a humidity chamber and acclimatized by gradually lowering the humidity over 2-3 weeks (temperature 20 ± 2 C, humidity 95%, PPF = 55 μmol m -2 s -1, 16 h photoperiod). Hardened-off plants can be maintained in the greenhouse (temperature 20 ± 2 C, humidity 85%, maximum PPF = 90 μmol m -2 s -1, 16 h photoperiod) (Debnath 2005, 2006). Rooting can be induced ex vitro with complete success (Hayashi et al. 1997; Borkowska et al. 1999; Borkowska 2001) by transferring microshoots directly in rockwool (Borkowska et al. 1999). After one month, the plantlets rooted ex vitro are planted in 1 peat : 1 rockwool (v/v) and grown in the greenhouse for acclimatization (Borkowska 2001). In vitro-derived strawberry shoots were rooted ex vitro in granulated, water-repelling and water absorbing mineral wool (Grodan) mixed in a proportion of 1:1 (v/v) by Sowik et al. (2003). Fertilizer (10% N, 52% P, 10% K) are applied daily at the rate of 250 mg/l during the first week, followed by 800 mg/l during the next three weeks with alternating every second day. Rooting is done at C under a PPFD of 75 µmol m -2 s -1. The rooted shoots are planted in sand, soil substrate and leaf compost (0.3:5:1.3, v/v) medium (Sowik et al. 2003). Debnath (2006) developed a protocol that enables strawberry micropropation in one step, i.e. multiplying shoots and having them rooted in the same culture medium. The use of microcuttings, giving both root and shoot growth in a medium containing cytokinin, is emerging as a better choice for micropropagation of strawberries than multiple shoot proliferation, (using a cytokinin supplemented medium) with subsequent rooting of shootlets. In vitroderived strawberry shoots can be proliferated, elongated and rooted on zeatin-containing medium. Zeatin alone at very low levels (1-2 μm) produced two to three shoots per explant, averaging 88% rooting incidence in a single medium in Bounty strawberry. Furthermore, the protocol did not use auxin in the culture medium, which lowers the cost and reduces the probability of somaclonal variation among 4

5 Strawberry culture in vitro. Debnath and Teixeira da Silva the proliferated plants. The main advantage of this protocol is that all the shoot tips of the in vitro-grown plantlets can be used for shoot proliferation and rooting, whereas basal rooted nodal segments can be transferred to the potting medium and acclimatized in the greenhouse. The protocol can eliminate stage II of micropropagation and can increase both multiplication rate and rooting rate; this translates into a faster micropropagation of strawberry. The technique is now routinely used at the Atlantic Cool Climate Crop Research Centre in St. John s, NL, Canada. Somatic embryogenesis Somatic embryogenesis involves the development of bipolar embryos from embryogenically-competent somatic cells in vitro. In contrast to organogenesis, where microshoots and roots develop on different media, somatic embryogenesis is apparently a one-step procedure involving the development of embryos having both a shoot and a root pole, as in the zygotic embryos. The initiation and development of embryos from somatic tissues was first observed by Steward et al. (1958) and Reinert (1958) in cultures of carrot tissues. Wang et al. (1984) reported somatic embryogenesis from strawberry cotyledons on MS medium supplemented with 22.6 µm 2,4-D, 2.2 µm BA and 500 mg/l casein hydrolysate where few of the embryogenic tissues developed into somatic embryos. Morphologically normal plants were obtained from somatic embryos that were transferred to MS medium containing 2.89 µm GA 3 or 2.22 µm BA µm NAA. Maintenance of the embryogenic cultures was, however, unsuccessful. Donnoli et al. (2001) reported somatic embryogenesis in 8% of the embryogenic calli in strawberry cultivar Clea on MS medium supplemented with 4.88 µm BA and 4.90 µm IBA. Somatic embryogenesis research with strawberries is still in a preliminary stage and some more efforts would be required to develop the technology (Graham 2005). Bioreactor micropropagation Automated bioreactors for large scale production of tissue culture plants are important for commercial success of the micropropagation industry. Bioreactors are self-contained, sterile environments which capitalize on liquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and control over microenvironmental conditions (aeration, agitation, dissolved oxygen, etc.) (Paek et al. 2005). The use of large-scale liquid cultures and automation has the potential to resolve the manual handling of the various stages of micropropagation and decreases production cost significantly. Bioreactor systems have been introduced for mass propagation of horticultural plants (Levin and Vasil 1989) and have proven their potential for large-scale micropropagation. Different types of bioreactors developed for optimal mixing of oxygen, nutrients and culture without severe shear stress are generally two types: (i) mechanically agitated bioreactors and (ii) pneumatically agitated and non-agitated bioreactors (Paek and Chakrabarty 2003). Culture in liquid medium is advantageous for several plant species but often causes asphyxia and hyperhydricity, resulting in malformed plants and loss of material. The malformations are manifested in glossy hyperhydrous leaves with distorted anatomy. To overcome these problems, two major solutions for malformation control includes: use of growth retardants to control rapid proliferation and temporary immersion bioreactors (TIB, Ziv et al. 2003) in which the explants are alternately exposed to liquid cultivation medium and air. To avoid cell clumping in cell suspension cultures of cell line FAR (Fragaria ananassa R), Edahiro and Seki (2006) suggested the inclusion of 0.1 mm L-α-aminooxy-β-phenylpropionic acid, or AOPP, a specific inhibitor of phenylalanine ammonia lyase the starting and key enzyme of the phenylpropanoid pathway. Limited reports are available on in vitro bioreactor strawberry culture (Takayama and Akita 1998). Hanhineva et al. (2005) reported shoot regeneration from leaf explants of five strawberry cultivars in a commercially available TIB bioreactors (RITA, VITROPIC, Saint-Mathieu-de- Tréviers, France) containing liquid MS medium with 9 µm TDZ and 2.5 µm IBA (Table 1). The TIB system proved to be well suited for shoot propagation and for subsequent subculture of the developing plantlets. Regeneration frequencies were 70 ± 8 to 94 ± 2% and 83 ± 5 to 92 ± 3% in the TIB system and on semi-solid medium, respectively. The labour time taken by the TIB system was less than half of the time required for handling plant material for cultivation on semi-solid medium. Field evaluation of micropropagated plants Increased branching and vigorous vegetative growth are often noted in plants produced through in vitro culture. Differences in performance of tissue-cultured and conventionally propagated plants have been investigated for strawberry (Swartz et al. 1981; Boxus et al. 1984; Cameron et al. 1989; Cameron and Hancock 1986; López-Arand et al. 1994; Szczygiel and Borkowska 1997; Szczygiel et al. 2002). Tissue culture-derived strawberry plants grow more vigorously producing more crowns and runners and increased petiole length, yield per area and number of inflorescences per crown than conventionally propagated plants (Swartz et al. 1981; Boxus et al. 1984; Cameron et al. 1989; López-Arand et al. 1994). Zebrowska et al. (2003) reported that in comparison to vegetatively propagated plants, Teresa strawberry microplants produced more leaves, runners and inflorescences. Also, yielding and resistance to leaf scorch of these plants were much higher. Litwińczuk (2004) compared strawberry plants of cv. Senga Sengana obtained in vitro from axillary and adventitious shoots with their runner progeny and with standard runner (control) plants under field conditions. In the planting year, in vitro obtained plants developed significantly more crowns and runners while compared to other groups. Such differences, especially in runners number were not observed in the next two years. In the planting year, all in vitro propagated plants and about 80% their runner progeny flowered contrary to control (the only 3% plants). Every year tissue culture plants developed significantly more inflorescences than other studied groups. Plants obtained in vitro produced bigger fruits and higher yield than other groups in the first two years. However, a reduction of berry yield for tissue culture plants in contrast with control was observed in third year only. The primary effects, increased vigour and axillary bud activity, are possibly related to the forced proliferation in vitro through hormonally induced crown branching (Swartz et al. 1981). Micropropagated strawberry Gorella showed higher resistance to frost damage than did standard runner plants, when injury was evaluated in the field in spring (Rancillac and Nourrisseau 1989). Similarly, Dalman and Malata (1997) found that micropropagated Senga Sengana strawberry plants overwintered better than did the plants produced from runners, although for Mari the opposite was observed, and for Jonsok no differences between the two types of plants occurred. Palonen and Lindén (2001) compared cold hardiness and overwintering of three types of strawberry plants of cultivars Senga Sengana and Jonsok : (i) micropropagated virus-free elite plants, (ii) certified plants (runner plants from elite plants) and (iii) ordinary plants (runner plants of conventionally propagated plants from a strawberry farm). No consistent differences in cold hardiness among the three types of plants were detected during winter. Field evaluation did not reveal any differences in their winter survival, either. Micropropagated plants flowered more freely than did the plants produced through runners. 5

6 Fruit, Vegetable and Cereal Science and Biotechnology 1(1), Global Science Books SOMACLONAL VARIATION Clonal fidelity is one of the main concerns in commercial micropropagation, true-to-type propagules and genetic stability are prerequisites for the application of strawberry propagation in vitro. The occurrence of variation in plants regenerated from in vitro cultures was named as somaclonal variation by Larkin and Scowcroft (1981) and has been reported for morphological and yield variation in micropropagated strawberries (Graham 2005). There are concerns about genetic changes resulting from strawberry micropropagation. Discrete morphological variants have been observed in micropropagated strawberry plants, e.g., leaf variegation consisting of a narrow white streak in the leaf blade (Swartz et al. 1981), chlorosis of the leaves (Swartz et al. 1981), and growth changes including dwarfs, compact trusses, lack of runner production, and female sterility (Swartz et al. 1981). Moore et al. (1991) observed variability among micropropagated subclones of Olympus which were most likely transient responses to the micropropagation environment, not genetic. Generally, micropropagated plants have greater vigor, runner production, and yields than runner-propagated plants (Swartz et al. 1981). However, not all cultivars exhibit a yield increase (Cameron et al. 1985). Genetic stability during micropropagation is controlled by numerous factors including genotype, presence of chimeral tissue, explant type and origin, media type, types and concentrations of growth regulators, culture conditions (temperature, light, etc.) and duration of culture (Graham 2005). Neither somatic embryogenesis nor shoot organogenesis is widely used in commercial strawberry micropropagation as adventitiously regenerated plants may give rise to somaclonal variation. Somaclonal variations can be distinguished by their morphological, biochemical, physiological and genetic characteristics. Molecular markers are powerful tools in genetic identification of somaclonal variation with greater precision and less effort than phenotypic and karyologic analysis. Although somaclonal variation is not desirable for commer-cial micropropagation, it is a valuable tool in plant breeding wherein variation in tissue culture-regenerated plants from somatic cells can be used in the development of crops with novel traits. By applying selection pressure during tissue culture it is possible to develop somaclones resistance to biotic and abiotic stress (Jain 2001). Somaclonal variation has been associated with changes in chromosome number and structure, deamplification and amplification of genes, transposable element activation and alteration in DNA methylation (Phillips et al. 1994). Strawberry somaclonal variatnts have been produced through in vitro culture and selection. Although phenotypic variants might occur, even among plants regenerated from meristem (Sansavini et al. 1989), it is generally emphasized that genetic variation is associated with regeneration from callus culture (Popescu et al. 1997). Popescu et al. (1997) observed useful variation in plant and fruit characteristics in strawberry plants regenerated from leaf and petiole-derived callus. Both genotype and type of explant strongly influence the occurrence of somaclonal variation. A variant having a modified (white) colour of flesh for all fruits was induced from petiolederived callus of cv. Gorella (Popescu et al. 1997). Some regenerants evidence reduced susceptibility to soil borne fungi causing plant wilting (Battistini and Rosati 1991; Toyoda et al. 1991). Variants for earliness, calyx separation, mildew susceptibility and ploidy level were also found (Simon et al. 1987). Sansavini et al. (1989) reported the chlorophyll-mutant white stripe, chlorosis and dwarfism, the variants most commonly associated with micropropagated strawberry. The seven cultivars tested showed differential susceptibility to these alterations and these somaclonal variations were easily transmitted to the runners but declined markedly over the growing season. Transmission also occurred sexually, the symptoms themselves being more marked when the mother plant was affected. In selfing, S 1 offspring had a 26.7% incidence of white stripe, 60% of dwarfism and semidwarf and 66.7% of chlorosis. In crossing of affected and normal plants, white stripe affected only 15.4% and dwarfism 56.3% of F 1 seedlings (Sansavini et al. 1989). Leaf chlorosis, white streak and dwarfism were also noted in Pajoro strawberry somaclones regenerated from anther culture (Faedi et al. 1993). Variations in callus and cell suspension growth rates and in isoenzyme patterns of acid phosphatase, peroxidase and glutamate dehydrogenase among the regenerants of strawberry cultivars were reported by Damiano et al. (1995). Brandizzi et al. (2001) found variation in DNA content among strawberry regenerants from callus cultures. These variations were, however, lost after transfer to the greenhouse in four of the five cultivars. A significant change in DNA methylation status was noticed in cryopreserved strawberry shoot-tips (Hao et al. 2002). IN VITRO SELECTION In vitro selection is a useful tool in identifying plants resistant or tolerant to stresses produced by phytotoxins from pathogens, herbicides, cold temperature, aluminium, manganese and salt toxicity (Chaleff 1983). Usually, cells are subjected to a suitable selection pressure in vitro to recover any variant lines that have developed resistance or tolerance to the stress followed by regeneration of plants from the selected cell. This approach presumes that tolerance or resistance operating at the unorganized cellular level can act, to some degree of effectiveness, in the whole plant. The trait can be transferred to other plants if the tolerance/resistance has a genetic basis. In strawberry, research was done to obtain plants resistant/tolerant to Alternaria alternata (Takahashi et al. 1992), Botritis cinerae (Orlando et al. 1997), Colletotrichum acutatum (Damiano et al. 1997; Hammerschlag et al. 2006), Fusarium oxysporum (Toyoda et al. 1991), Phytophthora cactorum (Maas et al. 1993; Sowik et al. 2001), P. fragariae (Maas et al. 1993), P. nicotianae var. parasitica (Amimoto 1992), Rhizoctonia fragariae (Orlando et al. 1997) and to Verticillum dahliae (Sowik et al. 2001, 2003). Hammerschlag et al. (2006) used an in vitro screening system to evaluate strawberry cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to Anthracnose (C. acutatum) isolate Goff (highly virulent). Regenerants with resistance were genotype specific, and the highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. Insufficient winter hardiness is one of the major constrains limiting strawberry production in cool climates. An in vitro screening technology was determined for cold resistant strawberry seedlings (Rugienius and Stanys 2001). Strawberry plants were regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons on the medium with 1.0 mg/l BA and 0.5 mg/l NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo with a strong correlation (r = 0.93) between cold resistance in vitro and in vivo. GENETIC TRANSFORMATION Genetic transformation of Fragaria has made notable progress. The primary focus has been on transformation using genes with potential for pest and herbicide resistance, cold tolerance and ripening. Transformation in diploid and octoploid strawberries has been well documented using different constructs in various genotypes (Table 2). The earliest report of Agrobacterium tumefaciens-mediated transformation 6

7 Strawberry culture in vitro. Debnath and Teixeira da Silva Table 2 Genetic transformation with plant regeneration in strawberry. Cultivar/genotype Explant Vector/strain Marker gene(s) 1 Functional gene/trait 2 Reference Rapella Leaf, petiole Agro/LBA 4404 nos/nptii -- James et al Redcoat Leaf Agro/M P90 nptii/gus -- Nehra et al. 1990a, 1990b Chandler Leaf Agro/LBA 4404 nptii/gus -- Barceló et al Chandler Leaf Agro/LBA 4404 nptii/gus -- Harpster et al Totem Leaf Agro/EHA 105 nptii/gus -- Mathews et al Diploid F. vesca Alpine accession FRA 197 Leaf, petiole Agro/LBA 4404 nptii/gus -- Haymes and Davis 1998 Chandler Leaf Agro/LBA 4404, Biolistic nptii/gus -- Cordero de Mesa et al Gariguette, Polka, line no Leaf Agro/AGL0 nptii/gus -- Schaart et al Chandler Leaf Agro/LBA 4404 nptii/gus njjs25 Jimenez-Bermudez et al Pajaro Leaf Agro/LBA 4404 nptii/gus -- Ricardo et al Joliette Stipules Agro/LBA 4404 nptii pcht28 Chalavi et al Induka, Elista Leaf Agro/LBA 4404 nptii/gus -- Gruchała et al Hecker, La Sans Rivale, diploid accessions (FRA197, FRA198) Leaf, petiole Agro/EHA 105 nptii/gus -- Zhao et al Toyonaka Anther calli Biolistic nptii LEA3 Wang et al Chambly Shoot Agro/GV 3101 nptii wcor410a Houde et al Tiogar Leaf Agro/LBA 4404 nos/nptii APF Khammuang et al Firework Leaf Agro/CBE 21 nptii thau II Schestibratov and Dolgov 2005 Anther Leaf Agro/LBA 4404 nptii FagpS Park et al F. vesca diploid accessions Leaf Agro/GV 3101, LBA 4404 Hygromycin/gfp -- Oosumi et al Pájaro Leaf Agro/LBA 4404 nptii ch5b, gln2, ap24 Vellicce et al gfp = green fluorescent protein gene, gus = glucuronidase gene, nos = nopaline synthase gene, nptii = neomycin phosphotransferase gene. 2 ap24 = thaumatin-like protein gene from Nicotiana tabacum, APF = antifreeze protein gene from Antarctic fish, ch5b = chitinase protein gene from Phaseolus vulgaris, FagpS = antisense cdna of ADP-glucose pyrophosphorylase (AGPase) small subunit, gln2 = glucanase protein gene from N. tabacum, LEA3 = late embryogenesis abundant protein gene frm barley, njjs25 = strawberry pectate lyase gene, pcht28 = Lycopersicon chilense chitinase gene, thau II = thaumatin II protein gene, wcor410a = wheat dehydrin gene. of strawberry were made by Jelenkovic et al. (1986) followed by James et al. (1990) and Nehra et al. (1990a, 1990b). Although a direct gene transfer method has been reported (Nyman and Wallin 1992; Wang et al. 2004), methods using leaf disks or crown sections as explants for A. tumefaciens-mediated transformation are generally employed (Jelenkovic et al. 1986; James et al. 1990; Nehra et al. 1990a, 1990b; El Mansouri et al. 1996; Haymes and Davis 1998; Barceló et al. 1998; Ricardo et al. 2003; Folta et al. 2006; Mezzetti and Constantini 2006). Although Abdal-Aziz et al. (2006) have cautioned that the use of Agrobacterium plasmids often results in the integration of non- T-DNA sequences (i.e. that lies outside T-DNA, in particular the trfa gene) in an average of 65.7% of transgenic plants (ranging from 40 to 90%). The availability of micropropagation and regeneration systems coupled with the susceptbility to infection by Agrobacterium species (Uratsu et al. 1991) makes the strawberry well suited for Agrobacterium-mediated genetic transformation studies using different constructs and various germplasm (Table 2). Agrobacterium-mediated transformation involves a co-cultivation of the Agrobacterium strain with the explants in an organogenic regeneration system on a nutrient medium containing BA or TDZ with an auxin (2,4-D, IAA) (Schaart et al. 2002; Zhao et al. 2004; Oosumi et al. 2006). Selection of regenerants occurred on medium with mg/l kanamycin (Schaart et al. 2002; Chalavi et al. 2003) or 4 mg/l hygromycin (Oosumi et al. 2006). Cefotaxime, carbenicillin and/or ticaricillin are used to control Agrobacterium contamination after inoculation. The transfer of gene is generally confirmed by Southern blot analysis (James et al. 1990). Cordero de Mesa et al. (2000, 2004) reported a transformation protocol where gold particles were coated with Agrobacterium cells and used to bombard Chandler strawberry leaf explants. Folta et al. (2006) developed a rapid regeneration and transformation system for genetic line Laboratory Festival #9 derived from selfpollination of a productive Florida cultivar, Strawberry Festival. An effective method for the production of transgenic plants from which selectable marker genes have been removed has been reported by Schaart et al. (2004). The system combines a chemically inducible recombinase activity and a bifunctional selection system that allows the production of marker-free transgenic strawberry plants. Wang et al. (2004) transformed Toyonaka strawberry calli from anthers by particle bombardment with plasmid pby520 containing late embryogenesis abundant protein gene, LEA3, from barley (Hordeum vulgare L.). The bombarded calli were selected on 10 mg/l phosphinothricin (PPT) containing medium where 15.4% regeneration was observed. Hybridization signals detected by DNA dot blot analysis indicated foreign gene integration into the strawberry genome. The transformation efficiency varies among cultivars and a given media formulation works well with a subset of cultivars. A transformation frequency of 0.95% has been reported for the cultivar Rapella (James et al. 1990), 6.5% for Red Coat (Nehra et al. 1990b) and 11% for Firework (Schestibratov and Dolgov 2005) in Agrobacterium-mediated transformation system. Combining A. tumefaciens infection and biolistic bombardment on transformation, Cordero de Mesa et al. (2000) reported a transformation frequency of 20.7% in Chandler strawberry. A number of risks are associated with strawberry transformation including escapes, formation of chimeric shoots (Mathews et al. 1998) and somaclonal variation. To minimize these risks, rapid shoot regeneration and stringent selection are required. Oosumi et al. (2006) used hygromycin instead of kanamycin to suppress the growth of untranformed cells, yet to allow efficient transgenic shoot formation and to minimize escapes and chimaeras. Transformation has improved strawberries for many traits. Resent reports present evidence that cultivated strawberries may be engineered with specific pathogenesis-related (PR) genes that can decrease the severity of strawberry grey mold caused by Botrytis cinerae (Schestibratov and Dolgov 2005; Vellicce et al. 2006). Genes conferring strawberry mild yellow edge virus-coat protein (SMYEV-CP) (Finstad and Martin 1995), cowpea trypsin inhibitor (CPTI) (Graham et al. 1995), have been transferred into strawberry. Strawberry has been transformed with a rice chitinase gene (RCC2), and the resulting plants exhibit improved resistance to powdery mildew, imparted by the fungus Sphaerotheca humuli (Asao et al. 1997). The cowpea protease trypsin inhibitor gene (CpTi) was introduced to a strawberry cultivar and the CPTI overexpressing plants exhibited significantly higher root mass than control plants (Graham et al. 2002). Enhanced resistance to Verticillium daliae, the causal agent of verticillum wilt, was obtained in plants overexpressing 7